Aerococcus urinae - A potent biofilm builder in endocarditis

Abstract

The diagnosis of infective endocarditis (IE) remains a challenge. One of the rare bacterial species recently associated with biofilms and negative cultures in infective endocarditis is Aerococcus urinae. Whether the low number of reported cases might be due to lack of awareness and misidentification, mainly as streptococci, is currently being discussed. To verify the relevance and biofilm potential of Aerococcus in endocarditis, we used fluorescence in situ hybridization to visualize the microorganisms within the heart valve tissue. We designed and optimized a specific FISH probe (AURI) for in situ visualization and identification of A. urinae in sections of heart valves from two IE patients whose 16S rRNA gene sequencing had deteced A. urinae. Both patients had a history of urinary tract infections. FISH visualized impressive in vivo grown biofilms in IE, thus confirming the potential of A. urinae as a biofilm pathogen. In both cases, FISH/PCR was the only method to unequivocally identify A. urinae as the only causative pathogen for IE. The specific FISH assay for A. urinae is now available for further application in research and diagnostics. A. urinae should be considered in endocarditis patients with a history of urinary tract infections. These findings support the biofilm potential of A. urinae as a virulence factor and are meant to raise the awareness of this pathogen.

Fig 1. Specific identification of Aerococcus urinae by FISH.

Fixed cultures of Aerococcus sanguinicola and A. urinae, as well as heart valve tissue were simultaneously hybridized with the probes EUB338 (FITC), AURI (Cy3) and NONEUB (Cy5). DAPI was used to stain nucleic acids. Each row shows an identical microscopic field with filter sets for FITC, Cy3, Cy5 and DAPI, respectively. AURI only detected A. urinae in the culture and the heart valve section, thus demonstrating specific hybridization. Scale bar 10 μm. Subsequently, the novel FISH probe AURI (Cy3-labeled) was applied to new sections of the heart valve samples simultaneously with the EUB338 (FITC-labeled); thus confirming A. urinae as the infectious agent of the IE in situ and demonstrating the biofilm potential of these bacteria (Figs 2 and 3).

Fig 2. FISH reveals impressive biofilms on the mitral heart valve from the first patient, identifying the infectious agent as Aerococcus urinae.

FISH on sections of the mitral valve tissue simultaneously using the probes AURI (Cy3-labeled), EUB338 (FITC-labeled), NONEUB (Cy5), and the nucleic acid stain DAPI (blue) identified the infectious agent in situ as A. urinae. We found impressive monospecies bacterial biofilms that were partly FISH-positive (yellow) and partly visible only with the nucleic acid stain DAPI (blue). The FISH signal corresponds to a high ribosome content and indicates bacterial activity at the time of surgery. A. Overview of the mitral valve tissue specimen showing impressive structures consistent with bacterial biofilms on the surface of the heart valve. B. Magnification of the inset marked in A shows distinct cells positive for the probes EUB338 and AURI, thus identifying the infectious agent in situ as A. urinae.

Fig 3. FISH reveals Aerococcus urinae infiltrating the aortic heart valve from the second patient.

FISH of the aortic valve using the universal probe EUB338 (FITC), AURI (Cy3), NONEUB (Cy5), and the nucleic acid stain DAPI identifies A. urinae in situ. A. Overview of the aortic valve tissue specimen displaying large areas consistent with bacterial biofilms. (B) Magnification of the inset marked in A shows FISH-positive cells for the probes EUB338 and AURI (yellow) as well as DAPI-only positive cells (blue) within the heart valve tissue. Note the destructive growth of A. urinae in the heart valve tissue.